CN112094778A - Weissella LAS1, preparation method and application thereof - Google Patents

Abstract

The invention discloses Weissella LAS1, which solves the problem that white spots are easy to appear in soybean paste fermentation due to overhigh tyrosine content. The invention provides Weissella LAS1, comprising the application of Weissella LAS1 in the preparation of soybean paste, wherein the Weissella LAS1 is added in the steps of making yeast and fermenting the soybean paste; the inoculation amount of the Weissella LAS1 in the starter propagation step for preparing the soybean paste is 102-105 finished starters/g; the inoculation amount of the Weissella LAS1 inoculated in the fermentation step of the preparation of the soybean paste is 102-; the starter propagation step for preparing the soybean paste also comprises the step of adding aspergillus oryzae KD 11. The Weissella LAS1 provided by the invention has the advantages of effectively degrading the glycine, inhibiting the generation of white spots in the soybean paste, improving the flavor of the soybean paste and the like.

Description

Weissella LAS1, preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to Weissella LAS1, a preparation method and application thereof.

Background

The soybean paste is a traditional fermented seasoning in China, is prepared by fermenting soybeans with microorganisms such as aspergillus oryzae and the like, has fresh and salty moderate mouthfeel, and is deeply loved by consumers. However, white precipitates are often formed on the surface of soybeans during the fermentation preparation of soybean paste, which affects the organoleptic properties of the product. The soybean paste white spot mainly contains tyrosine, which is formed by hydrolyzing protein by a terminal peptidase to generate a large amount of tyrosine. The solubility of tyrosine in water is about 0.45g/L, and the solubility of tyrosine is reduced along with the increase of salinity; in 18% saline, the solubility of tyrosine is only about 0.2g/L, so that tyrosine generated in the fermentation preparation process of the soybean paste is easy to crystallize and separate out. So far, no efficient method for eliminating white spots of soybean paste has appeared.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: tyrosine is easily separated out in the preparation of the soybean paste, and white precipitates, namely white spots, are generated; the Weissella LAS1 can effectively degrade the content of tyrosine and inhibit white spots in the preparation of soybean paste.

The invention is realized by the following technical scheme:

a Weissella LAS1, further preferably, the Weissella LAS1 was deposited at 26.04.2020 at the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the following deposition addresses: the No. 3 Hospital No.1 of West Chen West Lu of the Korean-Yang district of Beijing is classified and named Weissella mesenteroides, the Latin name is Weissella paramesenterides, and the preservation number is CGMCC No. 19738.

The weissella LAS1 strain has the following characteristics:

(1) the morphological characteristics of the strain are as follows: the Weissella LAS1 strain is milk white on a flat plate, the colony edge is smooth, and the Weissella LAS1 strain is spherical under a microscope;

(2) part of the physiological and biochemical characteristics of the strain: the Weissella LAS1 can utilize glucose, lactose, xylose, arabinose and sucrose, the lactic acid production performance of the strain is good, and the optimal growth temperature of the strain is 37 ℃.

The Weissella LAS1 can rapidly degrade tyrosine in high salt environment, inhibit white spots in soybean paste preparation, and simultaneously does not produce biogenic amine, thereby improving the flavor of soybean paste.

Further preferably, the Weissella LAS1 comprises preparation of Weissella LAS1, and the liquid culture medium of the Weissella LAS1 comprises 10-25% of soy sauce crude oil, 20-40g/L of glucose and 12-16g/L of edible salt by volume ratio; the Weissella LAS1 is prepared by static culturing in liquid medium of Weissella LAS1 at 35-37 deg.C for 5-10 days. The culture and extraction of Weissella LAS1 are found in the sauce mash, and the culture environment condition can ensure the activity of Weissella LAS1, so that the Weissella LAS1 is effectively extracted and inoculated.

Further preferably, the application of Weissella LAS1 is used for preparing soybean paste.

Further preferably, weissella LAS1 is added during the fermentation step of the soybean paste preparation, or weissella LAS1 is added simultaneously during the starter propagation step and the fermentation step of the soybean paste preparation. The application method of the Weissella LAS1 in the preparation of the soybean paste provided by the invention comprises the steps of inoculating the Weissella LAS1 in the fermentation step or in the fermentation step and the starter propagation step at the same time, and converting the generated tyrosine into other aromatic compounds through an Eschka way, so as to degrade the tyrosine content, improve the flavor quality of the soybean paste and eliminate the generation of white spots of the soybean paste.

Further preferably, Weissella LAS1 is inoculated into a starter propagation material in the starter propagation step of the soybean paste preparation, the inoculation time of the Weissella LAS1 is the initial time of the starter propagation step, and the inoculation amount of the Weissella LAS1 is 102-105 finished starters/g; the Weissella LAS1 is inoculated into fermentation materials in the fermentation step of the soybean paste preparation, the inoculation time of the Weissella LAS1 is 1d of the fermentation step, and the inoculation amount of the Weissella LAS1 is 102-104 koji/g. Proper amount of Weissella LAS1 is added in the starter propagation step and the fermentation step in the preparation of the soybean paste, so that the soybean paste can be promoted to be effectively fermented, tyrosine generated by fermentation can be effectively degraded, and biogenic amine is not generated, so that the aim of not generating white spots in the production process of the soybean paste is fulfilled, meanwhile, the flavor of the soybean paste can be improved, and the mellow flavor of the soybean paste is improved.

Further preferably, the Weissella LAS1 further comprises Aspergillus oryzae KD11 during the starter propagation step of the soybean paste preparation. The inoculated aspergillus oryzae KD11 can improve the fermentation efficiency of the soybean paste, so that the soybean paste is more fully fermented.

Preferably, in the starter propagation step for preparing the soybean paste, aspergillus oryzae KD11 is inoculated into a starter propagation material, the inoculation time of aspergillus oryzae KD11 is the initial time of the starter propagation step, and the inoculation amount of aspergillus oryzae KD11 is 0.05-0.1 per thousand in mass ratio based on the total dry weight of the starter propagation material; the Aspergillus oryzae KD11 is obtained by mutating AS 3.042, and is preserved in China general microbiological culture center with the preservation name of KD11 and the preservation number of CGMCC No. 19626. The aspergillus oryzae KD11 adopted by the invention can ensure the content of amino acid nitrogen in the soybean paste in the production and fermentation process of the soybean paste, promote the fermentation of the soybean paste and further improve the flavor and taste of the soybean paste. Further preferably, the concentration of the brine in the fermentation step of the preparation of the soybean paste is 20 to 23%; the volume ratio of koji formation to saline water in the fermentation step of the preparation of the soybean paste is 1: (1-2). The saline water is added into the soybean paste according to the proportion, so that the decomposition and synthesis of enzyme can be promoted, and the fermentation period of the soybean paste is ensured; the preparation of the brine concentration can improve the delicate flavor of the fermented soybean paste, and meanwhile, the soybean paste is not easy to generate the phenomenon of sour taste deterioration.

Further preferably, the preparation of the soybean paste comprises:

step 1: pretreating raw materials; mixing soybeans and water according to a set mass ratio, soaking, and draining for later use;

step 2: steaming and boiling; cooking the drained soybeans;

and step 3: mixing and stirring materials; uniformly mixing the cooled soybeans and the flour according to a set mass ratio;

and 4, step 4: preparing yeast; inoculating Aspergillus oryzae KD11 and Weissella LAS1 into mixture of semen glycines and flour, ventilating, making starter, and culturing to obtain finished starter;

and 5: fermenting; mixing the obtained starter with saline water at a given mass ratio, adding Weissella LAS1, and fermenting to obtain soybean paste.

The invention has the following advantages and beneficial effects:

1. the Weissella LAS1 provided by the invention is characterized in that the Weissella LAS1 is added in the starter propagation step and the fermentation step of the soybean paste preparation. In the starter propagation step of the soybean paste preparation, Weissella LAS1 is inoculated into starter propagation materials, the inoculation time of the Weissella LAS1 is the initial time of the starter propagation step, and the inoculation amount of the Weissella LAS1 is 102-105 finished starters/g; the Weissella LAS1 is inoculated into a fermentation material in the fermentation step of the soybean paste preparation, the inoculation time of the Weissella LAS1 is 1d in the fermentation step, and the inoculation amount of the Weissella LAS1 is 102-104 koji/g. The added Weissella LAS1 can effectively degrade tyrosine generated in the fermentation preparation of the soybean paste, thereby inhibiting white spots in the soybean paste, and meanwhile, the added Weissella LAS1 can also promote the generation of alcohol substances in the fermentation preparation of the soybean paste, and improve the flavor quality of the soybean paste;

2. the Weissella LAS1 provided by the invention comprises the preparation of Weissella LAS1, wherein a liquid culture medium of the Weissella LAS1 comprises 10-25% of soy sauce crude oil, 20-40g/L of glucose and 12-16g/L of edible salt by volume ratio; the Weissella LAS1 is prepared by static culture in Weissella LAS1 liquid medium at 35-37 deg.C for 5-10 days. The culture and extraction of the Weissella LAS1 are discovered and extracted from the sauce mash, and the culture environmental conditions can ensure the activity of the Weissella LAS1, so that the effective extraction and inoculation of the Weissella LAS1 are realized;

3. the starter propagation step for preparing the soybean paste also comprises the step of adding aspergillus oryzae KD11, wherein the access time of the aspergillus oryzae KD11 is the initial time of the starter propagation step, and the inoculation amount of the aspergillus oryzae KD11 is 0.05-0.1 per thousand in mass ratio based on the total dry weight of starter propagation materials. The added aspergillus oryzae KD11 can ensure the content of amino acid nitrogen in the soybean paste in the production and fermentation process of the soybean paste, promote the fermentation of the soybean paste and further improve the flavor and taste of the soybean paste.

4. The concentration of the saline water in the fermentation step of the preparation of the soybean paste is 20 to 23 percent; the ratio of the mass of koji in the fermentation step of the preparation of the soybean paste to the volume of saline water is 1: (1-2). The saline water added into the soybean paste according to the proportion can promote the decomposition and synthesis of enzyme and ensure the fermentation period of the soybean paste; the preparation of the brine concentration can improve the delicate flavor of the fermented soybean paste, and meanwhile, the soybean paste is not easy to generate the phenomenon of sour taste deterioration.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

The measuring method of each substance index comprises the following steps:

and (3) measuring phenethyl alcohol, furanone and 4-ethylguaiacol in the soy sauce by adopting a GC-MS analysis method:

GC was fitted to an Agilent DB-wax capillary chromatography column (30m 0.25mm 0.25 μm) as a separation column; temperature programming: the initial temperature is 40 ℃, the temperature is maintained for 5min, then the temperature is raised to 60 ℃ at the speed of 5 ℃/min, and then the temperature is raised to 230 ℃ at the speed of 10 ℃/min, and the temperature is maintained for 8 min. The temperature of the sample inlet is 230 ℃, no shunt is carried out, the carrier gas is helium, and the flow rate is 1.0 mL/min. An ionization mode: electron Ionization (EI); electron energy: 70 eV; the voltage is 350V; the temperature of a connecting port is 280 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the mass scanning range m/z is 33-450.

And (3) measuring the content of lactic acid in the soy sauce by adopting a high performance liquid chromatography:

filtering the sample solution with 0.22 μm microporous membrane, pretreating with Sep-Pak C18 column, and purifying with NaH₂PO₄Separating with high performance liquid chromatography (C18 chromatographic column) at 40 deg.C under 0.8ml/L with buffer solution (pH 2.7) as mobile phase, detecting with ultraviolet detector at 210nm, and determining organic acid content with peak height or peak area standard curve.

Determination of amino acids in soy sauce (see national standard GB 5009.124-2016):

and respectively injecting the mixed amino acid standard working solution and the sample determination solution into an amino acid analyzer in the same volume, and calculating the concentration of amino acid in the sample determination solution by peak area through an external standard method.

Example 1

The embodiment provides Weissella LAS1 which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation name of LAS1 and the preservation number of CGMCC NO. 19738;

the preparation method of the Weissella LAS1 comprises the following steps:

the liquid culture medium of the Weissella LAS1 comprises 10-25% of soy sauce crude oil, 20-40g/L of glucose and 12-16g/L of edible salt by volume ratio;

the Weissella LAS1 is prepared by standing culturing in liquid medium of Weissella LAS1 at 35-37 deg.C for 5-10 days.

Weissella LAS1 is able to convert tyrosine into other aromatic compounds via the ehrlich pathway, thereby reducing tyrosine content; determination of weissella LAS1 degradation of tyrosine: inoculating the slant activated Weissella LAS1 thallus into liquid culture medium containing tyrosine, standing at 37 deg.C for 14 days, and detecting tyrosine content at interval of 1 d.

The liquid culture medium of tyrosine comprises 5.0g/L of yeast extract powder, 10.0g/L of tryptone, 6.0g/L of monopotassium phosphate, 0.034g/L of ferrous sulfate, 0.575g/L of magnesium sulfate, 20.0g/L of glucose, 25.0g/L of sodium acetate, 2.0g/L of ammonium citrate, 0.12g/L of manganese sulfate, 15.0g/L of agar, 1.0g/L of sorbic acid and 0.35g/L of tyrosine, wherein the pH value of the liquid culture medium of tyrosine is 5.5.

The results of the tyrosine content measurements are shown in Table 1. As can be seen from Table 1, the amount of tyrosine remained after 14d fermentation; after 14d fermentation, complete degradation of tyrosine was achieved.

TABLE 1 determination of tyrosine degradation by Weissella LAS1

Fermentation time (d)	Tyrosine residual quantity (g/L)
		0	0.34
2	0.34
		4	0.33
6	0.26
		8	0.12
10	0.05
		12	0.02
14	0

By inoculating Weissella LAS1 to a culture medium containing tyrosine, and determining that Weissella LAS1 has the effect of effectively degrading the content of tyrosine from the change of the tyrosine content detected periodically, the tyrosine is converted into other aromatic compounds through an Eschka pathway by a microbial degradation method to reduce the content of tyrosine, so that the generation of white spots in the preparation of the soybean paste is inhibited.

Example 2

In the application of weissella LAS1 provided in example 1, the method steps of weissella LAS1 in the preparation of soybean paste are as follows:

step 1: pretreating raw materials; the mass of soybeans and the volume of water were measured as 1: 2.5, soaking for 10 hours, and draining for later use;

step 2: steaming and boiling; steaming the drained soybeans for 7min at the temperature of 121 ℃;

and step 3: mixing and stirring materials; cooling the soybeans and flour to 50 ℃, and mixing the soybeans and the flour according to the ratio of 1: 2, uniformly mixing;

and 5: preparing yeast; cooling the mixture of soybean and flour to 40 ℃, inoculating Aspergillus oryzae KD11, wherein the inoculating amount of Aspergillus oryzae is 0.075 per mill based on the total dry weight of the mixture of soybean and flour, ventilating at 30 ℃ for koji making, and culturing for 42h to obtain finished koji;

step 6: fermenting; mixing the prepared finished koji with 22% saline water according to the mass ratio of 1: 2, and adding Weissella LAS1 bacterial liquid with the inoculation amount of 10³Fermenting at 37 deg.C for 80 days to obtain soybean paste.

The culture method of the Weissella LAS1 bacterial liquid comprises the following steps: inoculating a ring of activated Weissella LAS1 into soy sauce culture medium, wherein the soy sauce culture medium comprises 20% of soy sauce crude oil, 3g/L of glucose and 16g/L of edible salt by volume ratio; standing soy sauce culture medium at 37 deg.C for 7 d.

The fermentation preparation of the soybean paste is completed by inoculating Weissella LAS1 in the fermentation step in the preparation of the soybean paste, and only the Weissella LAS1 is inoculated in the fermentation step, so that less white spots are generated in the soybean paste, and the tyrosine content is reduced.

Example 3

In the application of weissella LAS1 provided in example 1, the method steps of weissella LAS1 in the preparation of soybean paste are as follows:

step 1: pretreating raw materials; the mass of soybeans and the volume of water were measured as 1: 2.5, soaking for 10 hours, and draining for later use;

step 2: steaming and boiling; steaming the drained soybeans for 7min at the temperature of 121 ℃;

and step 3: mixing and stirring materials; cooling the soybeans and flour to 50 ℃, and mixing the soybeans and the flour according to the ratio of 1: 2, uniformly mixing;

and 4, step 4: preparing yeast; cooling the mixture of soybean and flour to 40 deg.C, and inoculating Aspergillus oryzae KD11 and Weissella LAS 1; inoculating Aspergillus oryzae at a mass ratio of 0.075 ‰ based on total dry weight of soybean and flour mixture, inoculating Weissella LAS1 at an inoculation amount of 103/g to obtain finished koji, ventilating at 30 deg.C, and culturing for 42 hr to obtain finished koji;

and 5: fermenting; mixing the prepared finished koji with 22% saline water according to the mass ratio of 1: 2, and adding Weissella LAS1 strain with the inoculation amount of 10³Fermenting at 37 deg.C for 80 days to obtain soybean paste.

The culture method of the Weissella LAS1 bacterial liquid comprises the following steps: inoculating a ring of freshly activated LAS1 to a soy sauce medium comprising 20% by volume soy sauce base oil, 3g/L glucose, 16g/L table salt; and (3) statically culturing the soy sauce culture medium at 37 ℃ for 7 d.

By inoculating Weissella LAS1 in the starter propagation step of soybean paste preparation and inoculating Weissella LAS1 in the fermentation step, the tyrosine content is obviously reduced, no visible white spots are generated in the soybean paste, the aims of effectively degrading tyrosine and inhibiting the generation of white spots of the soybean paste are fulfilled, the flavor of the soybean paste is improved, and the soybean paste has stronger mellow fragrance.

Example 4

This example differs from examples 2 and 3 in that the fermentation step was performed without access to Weissella LAS1 and Aspergillus oryzae KD11 and without access to Weissella LAS1 and the fermentation step was performed without access to Aspergillus oryzae AS 3.042, all other process steps and conditions being AS defined in example 3, and was used AS a blank control.

TABLE 2 fermentation index determination

As can be seen from table 2, the tyrosine content was greatly reduced in both examples 2 and 3, compared to example 4, i.e., the blank control, and example 4 had a large number of visually observable white spots, example 2 had a small number of visually observable white spots, whereas example 3 had no visually observable white spots, indicating that the effect of simultaneously inoculating weissella LAS1 in the koji-making step and the fermentation step was optimal. Meanwhile, the contents of the phenethyl alcohol, the 4-ethylguaiacol and the furanone in the embodiment 2 and the embodiment 3 are greatly improved compared with the embodiment 4 of the blank control group, wherein the contents of the phenethyl alcohol, the 4-ethylguaiacol and the furanone in the embodiment 3 are the highest, the mellow taste is strong, the sauce flavor is strong, and the blind evaluation is also the best, which shows that the inoculation of the Weissella LAS1 in the starter propagation step and the fermentation step can also promote the generation of alcohol substances in the soybean sauce and improve the flavor.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The Weissella LAS1 is characterized in that the Weissella LAS1 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation name of LAS1 and the preservation number of CGMCC NO. 19738.

2. A method of making Weissella LAS1 for use in making a Weissella LAS1 as claimed in claim 1,

the liquid culture medium of the Weissella LAS1 comprises 10-25% of soy sauce crude oil, 20-40g/L of glucose and 12-16g/L of edible salt by volume ratio;

the Weissella LAS1 is prepared by static culturing in liquid medium of Weissella LAS1 at 35-37 deg.C for 5-10 days.

3. Use of a weissella LAS1, said weissella LAS1 being a weissella LAS1 as claimed in claim 1 or a weissella LAS1 prepared by the method of making a weissella LAS1 as claimed in claim 2, for the preparation of soybean paste.

4. The use of Weissella LAS1 as claimed in claim 3 wherein Weissella LAS1 is added during both the starter propagation step and the fermentation step of the soy sauce preparation.

5. The use of Weissella LAS1 as claimed in claim 4 wherein said Weissella LAS1 is added to the starter propagation material during the starter propagation step of soybean paste preparation, the time of said Weissella LAS1 is the initial time of the starter propagation step and the amount of said Weissella LAS1 is 102-105/g starter propagation.

6. The use of the Weissella LAS1 as claimed in claim 4 wherein the Weissella LAS1 is added to the fermented material during the fermentation step of the soy sauce preparation, the Weissella LAS1 is added at 1 st day of the fermentation step and the Weissella LAS1 is added at 10 th day²-10⁴One/g finished koji.

7. The use of Weissella LAS1 as claimed in claim 3 wherein the koji preparation step further comprises the addition of Aspergillus oryzae KD 11.

8. The use of Weissella LAS1 as claimed in claim 7 wherein Aspergillus oryzae KD11 is inoculated into the koji making material during the koji making step of soybean paste preparation, the time of inoculation of Aspergillus oryzae KD11 is the initial time of the koji making step, the amount of inoculation of Aspergillus oryzae KD11 is 0.05% o to 0.1% o by mass based on the total dry weight of the koji making material; the Aspergillus oryzae KD11 is obtained by mutating AS 3.042, and is preserved in China general microbiological culture center with the preservation name of KD11 and the preservation number of CGMCC No. 19626.

9. The use of a Weissella LAS1 as claimed in claim 3 wherein the soy sauce is prepared with a brine concentration of 20% to 23% during the fermentation step of the soy sauce preparation; the ratio of the mass of the koji in the fermentation step of the preparation of the soybean paste to the volume of the saline water is 1: (1-2).

10. The use of a Weissella LAS1 as claimed in claim 3 wherein the preparation of said soybean paste comprises,

step 1: pretreating raw materials; mixing soybeans and water according to a set mass ratio, soaking, and draining for later use;

step 2: steaming and boiling; cooking the drained soybeans;

and step 3: mixing and stirring materials; uniformly mixing the cooled soybeans and the flour according to a set mass ratio;

and 4, step 4: preparing yeast; inoculating Aspergillus oryzae KD11 and Weissella LAS1 into mixture of semen glycines and flour, ventilating, making starter, and culturing to obtain finished starter;

and 5: fermenting; mixing the obtained starter with saline water at a given mass ratio, adding Weissella LAS1, and fermenting to obtain soybean paste.

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